The present invention relates to the field of extracellular signal regulated kinases (ERKs), which are also referred to as mitogen-activated protein (MAP) kinases.
None of the following discussion of the background of the invention is admitted to be prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine phosphatases (TPs) and tyrosine kinases (TKs), including receptor tyrosine kinases and non-receptor tyrosine kinases.
Receptor tyrosine kinases (RTKs) belong to a family of transmembrane proteins and have been implicated in cellular signaling pathways. The predominant biological activity of some RTKs is the stimulation of cell growth and proliferation, while other RTKs are involved in arresting growth and promoting differentiation. In some instances, a single tyrosine kinase can inhibit, or stimulate, cell proliferation depending on the cellular environment in which it is expressed.
RTKs are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. Ligand binding to membrane-bound receptors induces the formation of receptor dimers and allosteric changes that activate the intracellular kinase domains and result in the self-phosphorylation (autophosphorylation and/or transphosphorylation) of the receptor on tyrosine residues. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signaling molecules, thereby activating various signal transduction pathways.
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases do not contain a hydrophobic transmembrane domain or an extracellular domain and share non-catalytic. domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 domains and SH3 domains. The non-catalytic domains are thought to be important in the regulation of protein-protein interacions during signal transduction.
A central feature of signal transduction is the reversible phosphorylation of certain proteins. Receptor phosphorylation stimulates a physical association of the activated receptor with target molecules, which either are or are not phosphorylated. Some of the target molecules such as phospholipase Cxcex3 are in turn phosphorylated and activated. Such phosphorylation transmits a signal to the cytoplasm. Other target molecules are not phosphorylated, but assist in signal transmission by acting as adapter molecules for secondary signal transducer proteins. For example, receptor phosphorylation and the subsequent allosteric changes in the receptor recruit the Grb-2/SOS complex to the catalytic domain of the receptor where its proximity to the membrane allows it to activate ras. The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular signal transduction include Aaronson, Science, 254:1146-1153, 1991; Schlessinger, Trends Biochem. Sci., 13:443-447, 1988; and Ullrich and Schlessinger, Cell, 61:203-212, 1990.
A variety of mitogens as well as tumor promoters and agents which cause cellular differentiation can initiate a signalling cascade leading to phosphorylation and activation of mitogen-activated protein (MAP) kinases (also called ERKs). Boulton et al., Biochemistry 30:278-286(1991) and Boulton et al., Science 249:64-65 (1990) describe the purification and cloning of a MAP2/MBP kinase which they named extracellular signal-regulated kinase 1 (ERK-1). Using probes derived from ERK-1, two novel kinases were identified, ERK-2 and ERK-3 (Boulton and Cobb, Cell Regulation 2:357-371, 1991; Boulton et al., Cell 65:663-675, 1991). A fourth ERK has been briefly described (Cobb et al., Cell Regulation 2:965-978, 1991 and WO 91/19008 published Dec. 12, 1991). These proline-site-directed serine-threonine kinases in turn phosphorylate transcription factors such as p65TCF/Elk-1, c-jun and c-myc and thus appear to play a crucial role in signal transduction by converting extracellular stimuli into transcriptional activation.
The mechanism of activation of MAP kinases has been intensively investigated and revealed a conserved signalling cascade initiated by ligand induced activation of receptor tyrosine kinases which leads to a sequential activation of a series of protein kinases. The activated growth factor receptor signals via ras to the serine-threonine kinase raf which directly activates the MAP-kinase-ERK-kinase MEK. MAP kinases are stimulated by phophorylation on two regulatory threonine and tyrosine residues, respectively, which is catalyzed by activated MEK. Upon activation, MAP kinases have been reported to translocate into the nucleus and phosphorylate transcription factors. Another substrate of MAP kinases is the S6 kinase II (pp90 rsk) which is activated by ERKs and might then control protein translation. MAP kinases can also be activated by TPA which stimulates PKC and signals to MEK via raf. However, this pathway appears to be dependent on ras. A negative regulator of MAP kinase activity has been identified by cloning of a dual specificity phosphatase encoded by an immediate early gene (Charles et al. 1993, PNAS 90, 5292-5296) that seems to be highly specific for MAP kinases (Sun et al. 1993, Cell 75, 487-493).
One or more signal transduction pathways are belleived to control the differentiation of myoblasts. Upon depletion of serum growth factors from the culture medium at high cell density, proliferating skeletal myoblasts cease DNA synthesis, start to express muscle-specific genes (biochemical differentiation) and fuse to form multinucleate myotubes (terminal differentiation). However, upon terminal differentiation, myoblasts loose their ability to reenter the cell cycle in response to growth factor stimulation. This presumably involves the retinoblastoma gene product pRB and its interaction with members of the basic helix-loop-helix myogenic factor family.
While some of these muscle specific transcription factors, namely MyoD and myf5, are constitutively expressed both in cycling myoblasts as well as in myotubes, myogenin expression is induced when myoblasts start to differentiate. However, not only transcriptional regulation, but also posttranslational modification such as phosphorylation which has been reported for MyoD1 and myogenin as well as myf5 may influence commitment to myogenesis and maintenance of the differentiated state. Activated oncogenes like ras and src as well as growth factors which are involved in or initiate signal transduction, inhibit myogenesis. In addition, PKC is able to phosphorylate myogenin and could be a major mediator of this inhibition.
The present invention provides nucleic acid molecules coding for ERK-5; ERK-5 polypeptides; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antisense ERK-5 nucleic acid constructs; antibodies having binding affinity to an ERK-5 polypeptide; hybridomas containing the antibodies; nucleic acid probes for the detection of ERK-5 nucleic acid; a method of detecting ERK-5 nucleic acid or polypeptide in a sample; kits containing nucleic acid probes or antibodies; a method of detecting a compound capable of binding to ERK-5 or a fragment thereof; a method of detecting an agonist or antagonist of ERK-5 activity; a method of agonizing or antagonizing ERK-5 associated activity in a mammal; a method of treating diabetes mellitus, skeletal muscle diseases, Alzheimer""s disease, or peripheral neuropathies in a mammal with an agonist or antagonist of ERK-5 activity; and a pharmaceutical composition comprising an ERK-5 agonist or antagonist.
ERK-5 shows 6% similarity (38% identity) to the human ERK1 peptide sequence, 64% similarity to the rat ERK1 and ERK2 (39% and 37% identity, respectively) and 55% similarity to the rat ERK3 (30% identity). Featured herein are the molecular cloning and characterization of a novel member of the MAP kinase family, termed hERK5, from human skeletal muscle. The gene encodes a protein of 393 amino acids which delivers an apparent molecular weight of 45 kD. It was overexpressed in different eukaryotic cell systems without changing their growth rate. hERK5 can be activated by orthovanadate treatment of overexpressing cells, but not with PCS or growth factors. When overexpressed in the mouse myoblast cell line C2C12, hERK5, in contrast to its kinase negative mutant, increases terminal differentiation after serum withdrawal. These results indicate that hERK5 might play a role in commitment or differentiation of myoblasts, but not, like other members of the MAP kinase family, in cellular growth and proliferation
Thus, in one aspect, the present invention provides an isolated, enriched, or purified polypeptide comprising an amino acid sequence corresponding to at least 9 contiguous amino acids of SEQ. ID. NO. 2.
By xe2x80x9cisolatedxe2x80x9d in reference to a polypeptide is meant a polymer of 2 (preferably 9, more preferably 13, most preferably 25) or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is the predominate sequence present (at least 10-20% more than any other sequence) and is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no amino acid from other sources. The other source amino acid may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to elevate the proportion of the desired amino acid.
It is also advantageous for some purposes that an amino acid sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
In another aspect, the invention provides an organism or cell that contains a recombinant nucleic acid molecule comprising, 5xe2x80x2 to 3xe2x80x2, a promoter effective to initiate transcription in a host cell and a nucleic acid sequence encoding at least 9 contiguous amino acids of SEQ. ID. NO. 2. The present invention also provides an organism or cell that contains a recombinant nucleic acid molecule comprising a transcriptional region functional in a cell, a sequence complimentary to an RNA sequence encoding at least 9 amino acids of SEQ. ID. NO. 2, and a transcriptional termination region functional in said cell.
In another aspect the invention provides an antibody having a detectably stronger binding affinity to a polypeptide comprising at least 9 contiguous amino acids of SEQ. ID. NO. 2, than to a polypeptide comprising 9 or more contiguous amino acids of ERK-1, ERK-2, ERK-3, or ERK-4. Also provided is a hybridoma which produces such an antibody.
In yet another embodiment the invention features a method of detecting the presence or amount of a polypeptide comprising at least 9 contiguous amino acids of SEQ. ID. NO. 2 in a sample. The method involves contacting said sample with an antibody as described above, under conditions such that immunocomplexes form, and detecting the presence or amount of said antibody bound to said polypeptide.
A diagnostic kit for performing such a method is also provided. The kit contains a first container means containing the antibody as described above, and second container means containing a conjugate comprising a binding partner of said monoclonal antibody and a label.
A method of detecting a compound capable of binding to a polypeptide comprising at least 9 contiguous amino acids of SEQ. ID. NO. 2 is also provided. The method involves the steps of incubating the compound with said polypeptide and detecting the presence of the compound bound to said polypeptide.
A method of detecting an agonist or antagonist of ERK-5 activity comprising incubating cells that produce ERK-5 in the presence of a compound and detecting changes in the level of ERK-5 activity is also provided.
Also featured is a method of agonizing or antagonizing ERK-5 associated activity in a mammal, preferably one with a muscle differentiation disorder, comprising administering to said mammal an agonist or antagonist to ERK-5 in an amount sufficient to effect said agonism or antagonism.
Also provided is a pharmaceutical composition comprising an ERK-5 agonist or antagonist in an amount sufficient to alter ERK-5 associated activity, and a pharmaceutically acceptable diluent, carrier, or excipient.
In another aspect the invention features a method of screening potential agents useful for treatment of a disease or condition characterized by an abnormality in a signal transduction pathway that contains an interaction between a ERK-5 polypeptide and a natural binding partner (NBP). The method involves assaying potential agents for those able to promote or disrupt the interaction as an indication of a useful agent.
By xe2x80x9cscreeningxe2x80x9d is meant investigating an organism for the presence or absence of a property. The process may include measuring or detecting various properties, including the level of signal transduction and the level of interaction between a ERK-5 poplypeptide and a NBP.
By xe2x80x9cdisease or conditionxe2x80x9d is meant a state in an organism, e.g., a human, which is recognized as abnormal by members of the medical community. The disease or condition may be characterized by an abnormality in one or more signal transduction pathways in a cell, preferably a muscle cell, wherein one of the components of the signal transduction pathway is either a ERK-5 polypeptide or a NBP.
Specific diseases or disorders which might be treated or prevented, based upon the affected cells include muscle cell differentiation disorders such as muscular dystrophies. In preferred embodiments, the methods described herein involve identifying a patient in need of treatment. Those skilled in the art will recognize that various techniques may be used to identify such patients.
By xe2x80x9cabnormalityxe2x80x9d is meant an a level which is statistically different from the level observed in organisms not suffering from such a disease or condition and may be characterized as either an excess amount, intensity or duration of signal or a deficient amount, intensity or duration of signal. The abnormality in signal transduction may be realized as an abnormality in cell function, viability or differentiation state. We have determined that such abnormality in a pathway can be alleviated by action at the ERK-5:NBP interaction site in the pathway. An abnormal interaction level may also either be greater or less than the normal level and may impair the normal performance or function of the organism. Thus, it is also possible to screen for agents that will be useful for treating a disease or condition, characterized by an abnormality in the signal transduction pathway, by testing compounds for their ability to affect the interaction between a ERK-5 polypeptide and a NBP, since the complex formed by such interaction is part of the signal transduction pathway. However, the disease or condition may be characterized by an abnormality in the signal transduction pathway even if the level of interaction between the ERK-5 polypeptide and NBP is normal.
By xe2x80x9cinteractxe2x80x9d is meant any physical association between polypeptides, whether covalent or non-covalent. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. Examples of non-covalent bonds include electrostatic bonds, hydrogen bonds, and Van der Waals bonds. Furthermore, the interactions between polypeptides may either be direct or indirect. Thus, the association between two given polypeptides may be achieved with an intermediary agent, or several such agents, that connects the two proteins of interest (e.g., a ERK-5 polypeptide and a NBP). Another example of an indirect interaction is the independent production, stimulation, or inhibition of both a ERK-5 polypeptide and NBP by a regulatory agent. Depending upon the type of interaction present, various methods may be used to measure the level of interaction. For example, the strengths of covalent bonds are often measured in terms of the energy required to break a certain number of bonds (i.e., kcal/mol) Non-covalent interactions are often described as above, and also in terms of the distance between the interacting molecules. Indirect interactions may be described in a number of ways, including the number of intermediary agents involved, or the degree of control exercised over the ERK-5 polypeptide relative to the control exercised over the NBP.
By xe2x80x9cdisruptxe2x80x9d is meant that the interaction between the ERK-5 polypeptide and NBP is reduced either by preventing expression of the ERK-5 polypeptide, or by preventing expression of the NBP, or by specifically preventing interaction of the naturally synthesized proteins or by interfering with the interaction of the proteins.
By xe2x80x9cpromotexe2x80x9d is meant that the interaction between a ERK-5 polypeptide and NBP is increased either by increasing expression of a ERK-5 polypeptide, or by increasing expression of a NBP, or by decreasing the dephosphorylating activity of the corresponding regulatory TP (or other phosphatase acting on other phosphorylated signalling components) by promoting interaction of the ERK-5 polypeptide and NBP or by prolonging the duration of the interaction. Covalent binding can be promoted either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling polypeptides, such as an antibody, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimideesters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom 1984, J. Immunol. 133:1335-2549; Jansen, F. K., et al., 1982, Immunological Rev. 62:185-216; and Vitetta et al., supra).
By xe2x80x9cNBPxe2x80x9d is meant a natural binding partner of a ERK-5 polypeptide that naturally associates with a ERK-5 polypeptide. The structure (primary, secondary, or tertiary) of the particular natural binding partner will influence the particular type of interaction between the ERK-5 polypeptide and the natural binding partner. For example, if the natural binding partner comprises a sequence of amino acids complementary to the ERK-5 polypeptide, covalent bonding may be a possible interaction. Similarly, other structural characteristics may allow for other corresponding interactions. The interaction is not limited to particular. residues and specifically may involve phosphotyrosine, phosphoserine, or phosphothreonine residues. A broad range of sequences may be capable of interacting with ERK-5 polypeptides. Using techniques well known in the art, one may identify several natural binding partners for ERK-5 polypeptides.
By xe2x80x9csignal transduction pathwayxe2x80x9d is meant the sequence of events that involves the transmission of a message from an extracellular protein to the cytoplasm through a cell membrane. The signal ultimately will cause the cell to perform a particular function, for example, to uncontrollably proliferate and therefore cause cancer. Various mechanisms for the signal transduction pathway (Fry et al., Protein Science, 2:1785-1797, 1993) provide possible methods for measuring the amount or intensity of a given signal. Depending upon the particular disease associated with the abnormality in a signal transduction pathway, various symptoms may be detected. Those skilled in the art recognize those symptoms that are associated with the various other diseases described herein. Furthermore, since some adapter molecules recruit secondary signal transducer proteins towards the membrane, one measure of signal transduction is the concentration and localization of various proteins and complexes. In addition, conformational changes that are involved in the transmission of a signal may be observed using circular dichroism and fluorescence studies.
In another aspect the invention features a method of diagnosis of an organism for a disease or condition characterized by an abnormality in a signal transduction pathway that contains an interaction between a ERK-5 polypeptide and a NBP. The method involves detecting the level of interaction as an indication of said disease or condition.
By xe2x80x9corganismxe2x80x9d is meant any living creature. The term includes mammals, and specifically humans. Preferred organisms include mice, as the ability to treat or diagnose mice is often predictive of the ability to function in other organisms such as humans.
By xe2x80x9cdiagnosisxe2x80x9d is meant any method of identifying a symptom normally associated with a given disease or condition. Thus, an initial diagnosis may be conclusively established as correct by the use of additional confirmatory evidence such as the presence of other symptoms. Current classification of various diseases and conditions is constantly changing as more is learned about the mechanisms causing the diseases or conditions. Thus, the detection of an important symptom, such as the detection of an abnormal level of interaction between ERK-5 polypeptides and NBPs may form the basis to define and diagnose a newly named disease or condition. For example, conventional cancers are classified according to the presence of a particular set of symptoms. However, a subset of these symptoms may both be associated with an abnormality in a particular signalling pathway, such as the ras21 pathway and in the future these diseases may be reclassified as ras21 pathway diseases regardless of the particular symptoms observed.
Yet another aspect of the invention features a method for treatment of an organism having a disease or condition characterized by an abnormality in a signal transduction pathway. The signal transduction pathway contains an interaction between a ERK-5 polypeptide and a NBP and the method involves promoting or disrupting the interaction, including methods that target the ERK-5:NBP interaction directly, as well as methods that target other points along the pathway.
In preferred embodiments the disease or condition which is diagnosed or treated are those described above, the agent is a dominant negative mutant protein provided by gene therapy or other equivalent methods as described below and the agents is therapeutically effective and has an EC50 or IC50 as described below.
By xe2x80x9cdominant negative mutant proteinxe2x80x9d is meant a mutant protein that interferes with the normal signal transduction pathway. The dominant negative mutant protein contains the domain of interest (e.g., an ERK-5 polypeptide or a NBP), but has a mutation preventing proper signaling, for example by preventing binding of a second domain from the same protein. One example of a dominant negative protein is described in Millauer et al., Nature Feb. 10, 1994. The agent is preferably a peptide which blocks or promotes interaction of the ERK-5 polypeptide and the NBP. The peptide may be recombinant, purified, or placed in a pharmaceutically acceptable carrier or diluent.
An EC50 or IC50 of less than or equal to 100 xcexcM is preferable, and even more preferably less than or equal to 50 xcexcM, and most preferably less that or equal to 20 xcexcM. Such lower EC50""s or IC50""s are advantageous since they allow lower concentrations of molecules to be used in vivo or in vitro for therapy or diagnosis. The discovery of molecules with such low EC50""s and IC50""s enables the design and synthesis of additional molecules having similar potency and effectiveness. In addition, the molecule may have an EC50 or IC50 less than or equal to 100 xcexcM at a muscle cell.
By xe2x80x9ctherapeutically effective amountxe2x80x9d is meant an amount of a pharmaceutical composition having a therapeutically relevant effect. A therapeutically relevant effect relieves to some extent one or more symptoms of the disease or condition in the patient; or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or condition. Generally, a therapeutically effective amount is between about 1 nmole and 1 xcexcmole of the molecule, depending on its EC50 or IC50 and on the age and size of the patient, and the disease associated with the patient.
Further objects and advantages of the present invention will be clear from the description that follows.